Previously, we selected mutants of human rotavirus (HRV) serotypes of epidemiologic importance during successive serial passages in primary African green monkey kidney (AGMK) cells at progressively lower suboptimal temperatures (30 degrees Celsius, 28 degrees Celsius, and 26 degrees Celsius). These cold-adapted mutants include strain D (VP4:1A; VP7:1), DS-1 (VP4:1B; VP7:2), Wa x DS-1 (VP4:1A; VP7:2), Wa x P (VP4:1A; VP7:3) and Wa x ST3 (VP4:1A; VP7:4). One cold-adapted strain D has undergone phase I studies in human volunteers. We investigated the role of specific rotavirus gene(s) of the cold-adapted (30 degrees Celsius) strain D involved in conferring (i) the cold-adaptation (ca) phenotype, i.e., capacity to replicate efficiently in vitro at 30 degrees Celsius, a temperature restrictive for wild-type (wt) virus, and (ii) the temperature sensitivity (ts) phenotype, i.e., restriction of replication in cell culture at 39 degrees Celsius in contrast to wt HRV which replicated efficiently at temperatures at 39 degrees Celsius. To identify the gene(s) associated with the ca and ts phenotypes, we generated reassortants by co-infection in AGMK or MA104 cells of a ca (30 degrees Celsius) and ts mutant of HRV strain D and wt HRV strain DS-1 (non-cold-adapted, non-temperature sensitive) at 33 degrees Celsius, a temperature permissive for both parental viruses. Fifty distinct reassortants were selected and plaque-purified at 33 degrees Celsius, the temperature used to generate the reassortants. The parental origin of each rotavirus gene segment was assigned for each reassortant by polyacrylamide gel electrophoresis. The efficiency of plaque formation of parental and reassortant viruses in MA104 cells at 30, 37, and 39 degrees Celsius will be determined.